Streptococcus pneumoniae is an important human pathogen responsible for pneumonia, meningitis and other invasive diseases throughout the world (ref. 1. Throughout this application, various references are referred to in parenthesis to more fully describe the state of the art to which this invention pertains. Full bibliographic information for each citation is found at the end of the specification, immediately preceding the claims. The disclosures of these references are hereby incorporated by reference into the present disclosure). S. pneumoniae is also one of the major three organisms which cause otitis media in infants and children (refs. 2, 3). Otitis media is the most common illness of early childhood with approximately 70% of all children suffering at least one bout of otitis media before the age of seven. Chronic otitis media can lead to hearing, speech and cognitive impairment in children. It is caused by bacterial infection with S. pneumoniae (approximately 50%), non-typeable H. influenzae (approximately 30%) and Moraxella (Branhamella) catarrhalis (approximately 20%). In the United States alone, treatment of otitis media costs between one and two billion dollars per year for antibiotics and surgical procedures, such as tonsillectomies, adenoidectomies and insertion of tympanostomy tubes. Because otitis media occurs at a time in life when language skills are developing at a rapid pace, developmental disabilities specifically related to learning and auditory perception have been documented in youngsters with frequent otitis media.
S. pneumoniae is a Gram-positive encapsulated coccus that usually grows in pairs or short chains. The capsules comprise complex polysaccharides and are the basis for dividing pneumococci into different serotypes. S. pneumoniae exposed to type-specific antiserum show a positive capsular precipiting reaction, termed the Neufeld quellung reaction, and 84 serotypes have been identified by this means.
A polyvalent pneumococcus vaccine was developed for preventing pneumonia and other invasive diseases due to S. pneumoniae in the adult and aging populations. The vaccine contains capsular polysaccharides (CPs) from 23 serotypes of S. pneumoniae. These CPs are T-cell-independent antigens. They stimulate mainly immunoglobulin M (IgM) antibody with weak memory and readily induce tolerance. Although anticapsular antibodies to S. pneumoniae have long been recognized as protective in adult and immunocompetent individuals, children under 2 years of age and immunocompromised individuals, including the elderly, do not respond well to T-cell independent antigens and, therefore, are not afforded optimal protection by the current pneumococcal vaccines (ref. 4). There is thus a need to improve the current 23-valent pneumococcus vaccine, in order to provide protection for infants and individuals with reduced immuno-responsiveness.
Pneumococcus type 14 is one of the types isolated most frequently from patients of all ages (ref. 8). Pn14 is neutral and is composed of the following repeating tetrasaccharide: ##STR1##
Pn14 is a comparatively poor immunogen among the pneumococcal capsular polysaccharides. In adults, it elicits only a fourfold rise in antibodies in .about.80% of vaccinees. This property may be the reason why type 14 pneumococcus is one of the most common types isolated from adult patients immunized with the polyvalent pneumococcal vaccine (ref. 9). Pn14 does not elicit protective levels of antibodies in infants and young children.
CP of Pn6 is another relatively poor antigen in the 23-valent vaccine. Pn6 CP fails to elicit protective levels of antibodies in children up to about 24 months of age and further immunization does not induce a booster response. There are two serotypes of S. pneumoniae Pn6, type 6A and type 6B, and the structure of types 6A and 6B pneumococcal polysaccharides differs only in the position of linkage of their .alpha.-L-rhamnopyranosyl residues to D-ribitol, which is critical to their relative stabilities. The structures of 6A and 6B polysaccharide are as follows:
Type 6A: .fwdarw.2).alpha.-D-Galp(1.fwdarw.3)-.alpha.-D-Glcp(1.fwdarw.3).alpha.-L-R hap(1.fwdarw.3)-D-ribitol-5-PO.sub.4 - PA0 Type 6B: .fwdarw.2).alpha.-D-Galp(1.fwdarw.3)-.alpha.-D-Glcp(1.fwdarw.3).alpha.-L-R hap(1.fwdarw.4)-D-Ribitol-5-PO.sub.4 - PA0 Type 1: EQU .fwdarw.3).alpha.-AATp(1.fwdarw.4).alpha.D-GalpA(1.fwdarw.3).alpha.DGalpA(1 .fwdarw. PA0 Type 2: ##STR2## PA0 Type 3: EQU .fwdarw.4).beta.D-Glcp(1.fwdarw.3).beta.D-GlcpA(1.fwdarw. PA0 Type 4: ##STR3## PA0 Type 5: ##STR4## PA0 Type 6A: ##STR5## PA0 Type 6B: ##STR6## PA0 Type 7F: ##STR7## PA0 Type 8: EQU .fwdarw.4).beta.D-GlcpA(1.fwdarw.4).beta.D-Glcp(1.fwdarw.4).alpha.D-Glcp(1. fwdarw.4).alpha.D-Galp(1.fwdarw. PA0 Type 9N: EQU .fwdarw.4).alpha.D-GlcpA(1.fwdarw.3).alpha.D-Glcp(1.fwdarw.3).beta.D-ManpNA c(1.fwdarw.4).beta.D-Glcp(1.fwdarw.4).alpha.D-GlcpNAc(1.fwdarw. PA0 Type 9V: ##STR8## PA0 Type 10A: ##STR9## PA0 Type 11A: ##STR10## PA0 Type 12F: ##STR11## PA0 Type 14: ##STR12## PA0 Type 15B: ##STR13## PA0 Type 17F: ##STR14## PA0 Type 18C: ##STR15## PA0 Type 19A: ##STR16## PA0 Type 19F: ##STR17## PA0 Type 20: ##STR18## PA0 Type 22F: ##STR19## PA0 Type 23F: ##STR20## PA0 Type 25F: PA0 Type 33F: ##STR21##
T-cell dependent properties have been conferred to the CP of pneumococcus type 6B (Pn6B) and its immunogenicity improved by covalently coupling it to tetanus toxoid (TT) (ref. 5). CP of pneumococcus type 14 (Pn14) has been conjugated to pertussis toxin (PT) (ref. 6, WO 94/04195). The Pn14-PT conjugate elicited antibodies to Pn14 in mice at levels estimated to be protective in humans and elicited PT neutralizing anti-PT antibodies. Fattom et al. (ref. 7) synthesized conjugates composed of CP from pneumococcus type 12 (Pn12) coupled to diphtheria toxoid (DT). These Pn12-DT conjugates were shown to be more immunogenic than the Pn12 CP alone in adult volunteers.
Diphtheria and tetanus toxoids are frequently used as carriers for poorly immunogenic antigens to produce conjugates. Repeated immunization with these toxoids will produce very high antibody titres to the toxoids which may be disadvantageous. It would be advantageous, therefore, to provide a different carrier for poorly immunogenic carbohydrate antigens, of which there are about six to ten of medical interest. It is also desirable to use a carrier which generates a protective immune response including antibodies that are neutralizing for a further target organism.
P6 is a 16 kDa outer membrane protein (OMP) from H. influenzae which constitutes 1 to 5% of the OMP content. The protein is modified by fatty acylation and appears to be analogous to the low-molecular-weight peptidoglycan associated lipoproteins found in other Gram-negative bacteria (refs. 10, 11, 12). P6 has been shown to be present in every non-typeable and typeable H. influenzae isolate and is highly conserved (ref. 13). P6 is surface-exposed and is a target for bactericidal human antibodies (ref. 14). Furthermore, antibodies raised against P6 protein provided protection in the infant rat model of bacteremia (refs. 15, 16). These features made P6 a vaccine candidate against meningitis and/or otitis media caused by H. influenzae.